The generation and maintenance of a B cell repertoire that is tolerant to self rely on multiple mechanisms that regulate the differentiation of autoreactive B cells. These mechansims are controlled by the nature and duration of interaction between B cell receptor (BCR) and its antigen. Multivalent membrane-bound self antigens or double-stranded DNA causes immature self-reactive B cells to undergo deletion or receptor editing. Lower valency antigens, such as a soluble antigen or single-stranded DNA, induce anergy. Anergy is characterized by a profound block in proximal antigen receptor signaling resulting in an antigen-unresponsive state. The induction and maintenance of anergy require chronic binding of antigen and signal transduction. Therefore, the strength of BCR signals dictates the fate of developing B cells. However, the molecular events that translate BCR signal strength to fate choice in tolerance induction and underlying signaling pathways are not well understood. We propose to map these signaling pathways genetically using a genome-wide shRNA screen. The rationale of our approach is that induction of a specific form of B cell tolerance will be defective when a component of the underlying signaling pathway is reduced by shRNA knockdown. As in a genetic screen, cells having desired phenotypic changes are first selected after they are transduced with a retroviral shRNA library. Then the shRNAs carried by these cells are amplified and sequenced resulting in the identification of underlying genes. Since it is technically challenging to carry out shRNA screen in vivo, we will first carry out the screen on WEHI-231 B cell lymphoma cell line. Crosslinking of BCR on WEHI-231 cells results in their apoptosis and it has been used as a model of tolerance induction. Our specific aims are i) to identify novel molecules of BCR signaling pathways in WEHI-231 cells; and ii) to establish the roles of identified genes in different in vivo models of B cell tolerance including clonal deletion, receptor editing and anergy. The goal of this project is to identify new components of differential BCR signaling pathways in tolerance induction. Mutations that affect BCR signaling can alter B cell fate decisions and result in autoimmune diseases. Defective tolerance induction has been demonstrated in both animal models and patients of autoimmune diseases. These studies should provide a refined signaling map which will be essential in dissecting the defects of B cell tolerance in autoimmune diseases. Such knowledge should offer new opportunities to develop novel immunomodulatory drugs. PUBLIC HEALTH RELEVANCE: Using a genetic approach, we propose to screen and identify new genes in signaling pathways that are important for establishing or maintaining tolerance in B cells. This refined signaling map will be essential to dissect tolerance defects in autoimmune diseases and to develop novel immunomodulatory drugs.